Food and Chemical Toxicology 65 (2014) 364–373

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Food and Chemical Toxicology

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House dust mite (Der p 10) and crustacean allergic patients may reactto food containing Yellow mealworm proteins

0278-6915/$ - see front matter � 2014 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.fct.2013.12.049

Abbreviations: BAT, basophil activation test; HDM, House dust mite.⇑ Corresponding author. Address: TNO, Utrechtseweg 48, 3704 HE Zeist, The

Netherlands. Tel.: +31 888665136.E-mail address: kitty.verhoeckx@tno.nl (K.C.M. Verhoeckx).

1 These authors contributed equally to the study.

Kitty C.M. Verhoeckx a,b,c,⇑,1, Sarah van Broekhoven d,1, Constance F. den Hartog-Jager b,c, Marco Gaspari f,Govardus A.H. de Jong a, Harry J. Wichers e, Els van Hoffen b,c, Geert F. Houben a,b,c, André C. Knulst b,c

a TNO, Zeist, The Netherlandsb Department of Dermatology/Allergology, University Medical Center Utrecht (UMCU), Utrecht, The Netherlandsc Utrecht Center for Food Allergy (UCFA), Utrecht, The Netherlandsd Laboratory of Entomology, Wageningen University and Research Centre, Wageningen, The Netherlandse Agrotechnology and Food Innovations, Wageningen University and Research Centre, Wageningen, The Netherlandsf Proteomics@UMG, Department of Experimental and Clinical Medicine, University ‘‘Magna Græcia’’ of Catanzaro, Catanzaro, Italy

a r t i c l e i n f o a b s t r a c t

Article history:Received 21 August 2013Accepted 30 December 2013Available online 9 January 2014

Keywords:Food allergyRisk assessmentYellow mealwormCross-reactivityCrustaceansHouse dust mite

Scope: Due to the imminent growth of the world population, shortage of protein sources for human con-sumption will arise in the near future. Alternative and sustainable protein sources (e.g. insects) are beingexplored for the production of food and feed. In this project, the safety of Yellow mealworms (Tenebriomolitor L.) for human consumption was tested using approaches as advised by the European Food SafetyAuthority for allergenicity risk assessment.Methods and results: Different Yellow mealworm protein fractions were prepared, characterised, andtested for cross-reactivity using sera from patients with an inhalation or food allergy to biologicallyrelated species (House dust mite (HDM) and crustaceans) by immunoblotting and basophil activation.Furthermore, the stability was investigated using an in vitro pepsin digestion test. IgE from HDM- andcrustacean allergic patients cross-reacted with Yellow mealworm proteins. This cross-reactivity wasfunctional, as shown by the induction of basophil activation. The major cross-reactive proteins were iden-tified as tropomyosin and arginine kinase, which are well known allergens in arthropods. These proteinswere moderately stable in the pepsin stability test.Conclusion: Based on these cross-reactivity studies, there is a realistic possibility that HDM- and crusta-cean allergic patients may react to food containing Yellow mealworm proteins.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction Council of 27 January 1997, concerning novel foods and novel food

Due to the imminent growth of the world population andincreasingly more demanding consumers (Tilman et al., 2011),shortage of protein sources for human consumption is to beexpected in the near future (Van Huis, 2013). Because availableenergy supplies, clean water and land are declining, new initiativesare initiated to find more sustainable protein sources thanconventional meat for the food and feed industry, including theproduction of mini-livestock such as edible insects (FAO, 2013;Van Huis, 2013).

The introduction of new food sources is regulated by theRegulation (EC) No 258/97 of the European Parliament and of the

ingredients. All food and food products that were not commonlyused for consumption before 1997 must be labelled as novel foods(http://eur-lex.europa.eu/). For new food products, it is importantto assess food safety in terms of microbial, nutritional, toxicologi-cal, and allergenic risks.

Food allergy is a major health concern in the Western society.The prevalence of food allergy is around 3–4% in the general pop-ulation (Sicherer, 2011). Symptoms of food allergy range from oralallergy to anaphylactic shock. In addition to direct sensitisation toproteins in foods, food allergy can also result from IgE cross-reactivity between proteins in other food products or inhalantallergens from other species. Cross-reactivity is well-known forvarious related proteins (e.g. vicilins, PR-10 proteins, tropomyo-sins) (Radauer et al., 2008).

New proteins and genetically modified foods are currently as-sessed for their allergenic potential using an allergenicity assess-ment strategy (decision tree) advised by the Food andAgriculture Organisation (FAO) and the World Health Organization

K.C.M. Verhoeckx et al. / Food and Chemical Toxicology 65 (2014) 364–373 365

(WHO) (FAO/WHO, 2001) or the weight of evidence approach de-scribed by the European Food Safety Authority (EFSA, 2010). Bothapproaches focus on the source of the gene, the similarity of theamino acids sequence of the new protein with that of known aller-gens, cross-reactivity with human sera from food allergic patients,and the stability of the protein tested in a static in vitro digestionmodel with pepsin.

In this study, the allergenicity of Yellow mealworm (Tenebriomolitor) protein was assessed. The mealworm is the larval stageof the Yellow mealworm beetle and is commercially produced asfeed for animals such as fish, reptiles and birds. It could addition-ally be considered an alternative protein source for humans (VanHuis, 2013). To our knowledge, little is known about the allergenicpotency of Yellow mealworm proteins. A number of publicationsreport occupational allergy in sensitised individuals, with symp-toms such as asthma, rhinoconjunctivitis, and contact urticaria.These reactions were mostly observed in people frequently work-ing with Yellow mealworm (Bernstein et al., 1983; Schroeckensteinet al., 1990). Only one publication reported anaphylaxis followingthe ingestion of Yellow mealworm by an individual with a knowninhalant allergy to this insect (Freye et al., 1996). A few cases havebeen described of allergic reactions upon the consumption of otherinsect species, including Mopane worms (Imbrasia belina) (Okezieet al., 2010) and Domestic silkworm pupae (Bombyx mori) (Liuet al., 2009).

Because Yellow mealworm may be introduced as food for hu-man consumption, the safety with respect to the potential devel-opment of food allergy needs to be assessed. In this study, theweight of evidence approach was used to assess the allergenic po-tency of Yellow mealworm proteins.

2. Materials and methods

2.1. Materials

All reagents were obtained from Sigma (St-Louis, USA), unless stated otherwise.Yellow mealworm in final larval instars was obtained from insect producing com-pany Kreca (Ermelo, the Netherlands).

2.2. Patient sera

Sera were collected at the University Medical Centre Utrecht (Utrecht, the Neth-erlands) from well characterised allergic patients that were positive for specific IgEmeasured by ImmunCAP ISAC (Immuno Solid-Phase Allergen Chip). In this study,sera were used from seven patients allergic to crustaceans and House dust mite(HDM) Der p 10. Negative control sera were used from patients (in total 15 patients)allergic to grass pollen, peanuts, fish or eggs and/or milk. These patients were notallergic to crustaceans or HDM Der p 10. This study was approved by the ethicalcommittee of the University Medical Centre Utrecht.

2.3. Preparation of Yellow mealworm extracts

Larvae were killed by freezing at �20 �C. Ten grams of frozen larvae were dis-rupted in 70 ml ice cold extraction buffer (20 mM Tris buffer pH 7.6, 1 mM phenyl-thiocarbamide and Halt Protease Inhibitor Cocktail (Pierce Protein BiologyProducts) using an ultra-turrax. After centrifugation (10 min 16000g at 4 �C), thesupernatant was split into two fractions. One fraction was frozen immediately(SRN1) while the other was dialysed overnight (SRN2) against cold 20 mM Tris buf-fer pH 7.6 using a 3500 Da dialysis membrane. Both fractions were stored at �20 �C.

The water insoluble residue was washed twice with extraction buffer followedby overnight extraction at 4 �C in 70 ml 6 M urea in extraction buffer (SRN3). Thesample was centrifuged (10 min 16000g at 4 �C) and the supernatant was storedat �20 �C. The protein concentration was measured using the Bradford method(Bio-rad).

2.4. Identification of proteins in Yellow mealworm extract

The three individual extracts (380 lg protein) were digested overnight withtrypsin (protein:trypsin 380:7.5) after reduction and alkylation according to thestandard digestion protocol (see Section 2.8). Digestion was confirmed by Coomas-sie-stained SDS–PAGE. Prior to LC-MS analysis, samples were dried using a vacuumconcentrator (MAXI Dry Plus, Heto-Holten, Denmark) and reconstituted in 0.1%

formic acid (FA) in water. Ten microlitre of extract (20 lg protein) was injectedon an Atlantis� dc18 column (1.0 � 150 mm, 3 lm, Waters) using gradient elutionwith a constant flow of 50 ll min�1. The gradient started with 95% A (0.1% v/v FA inwater) for 5 min followed by a linear increase to 45% B (0.1% v/v FA in MeCN) whichwas achieved in 25 min. This was followed by a linear increase to 95% B in 5 minand was kept at this gradient for another 5 min. The LC-MS system consisted of aThermo Orbitrap mass spectrometer (Breda, The Netherlands) coupled to a ThermoAccela auto sampler and pump. The electrospray interface was used in positive-ionmode and an ion-spray voltage of 4.5 kV was applied. Capillary temperature was setto 350 �C. The orbitrap was operated in data-dependent mode, selecting the top 5ions for MS/MS scans at 35% collision energy units. See for protein identification2.8.3.

2.5. Determination of allergic potential

The allergic potential of proteins identified in Yellow mealworm extracts ofwhich the sequence is known, as well as that of Yellow mealworm proteins witha known sequence present in UniProt database, was predicted using Allermatch™(http://www.allermatch.org). Comparison in Allermatch™ was based on UniProtas well as the WHO–IUIS database. An 80 amino acid sliding window alignmentwas performed with a 35% cut-off percentage (Fiers et al., 2004).

2.6. SDS–PAGE and immunoblotting

All three individual Yellow mealworm protein extracts (3 lg) or digests thereofwere diluted with sample buffer (50 mM Tris pH 6.8, 2% SDS, 10% glycerol, 2%b-mercaptoethanol) and analysed using 15% acrylamide/Tris–HCl gels (Criterion,Biorad, Germany). Control samples (extracts of shrimp/lobster, peanut, grass pollen,herring/cod, egg from ALK, Denmark) and a Protein marker (Bio-Rad) were run oneach gel. Proteins were visualised using Coomassie gel staining (Expedion, UK) orused for immunoblotting. For immunoblotting, proteins were transferred to a poly-vinylidene difluoride membrane (Bio-Rad). Membranes were blocked with 4% (w/v)Protifar (Nutricia, The Netherlands) in PBS/0.1% Tween 20 for 60 min after whichthey were incubated for 1 h with diluted patient sera (dilution depended on IgE ti-ters and ranged from 1:25 to 1:200) at room temperature. Bound IgE was detectedwith 1:30,000 diluted peroxidase-conjugated goat anti-human IgE (KPL, USA). Visu-alisation was performed using a chemiluminescent peroxidase substrate kit andblots were scanned using a Chemidoc XRS+ image scanner with Imagelab software(Bio-Rad). Bands of interest were excised from gel for identification of cross-react-ing proteins.

2.7. Immunoprecipitation

For immunoprecipitation, Dynabeads M-280 Tosylactivated (10 mg, Invitrogen)were used according to the manufacturer’s protocol. Beads were coated with 0.2 mgGoat anti-Hu IgE (Invitrogen). Conjugated beads were incubated for 1 h at 37 �Cwith 1 ml of a mixture of patient sera (bead 1) from patients 2, 5 and 6 (2:3:3) or1 ml serum from patient 1 (bead 2). Conjugated beads were cross-linked with5 mM BS3 (Pierce) according to the manufacturer’s protocol to ensure reusabilityof the beads. The beads were washed three times with 0.1% Tween 20 in PBS pH7.4, followed by overnight incubation at 37 �C with either 100 ll of a mixture ofSRN1 + SRN2 with bead 1 or 100 ll SRN 3 (500 lg protein) with bead 2. Proteinswere eluted with 2 times 100 ll 0.1 M glycine and the pH of the solution was neu-tralized using 20 ll of 1 M tris–HCl pH 8.5. Incubation with Yellow mealworm ex-tract was repeated twice and all elutates were pooled, freeze dried and stored at�80 �C until further analysis.

2.8. Identification of cross-reactive proteins

2.8.1. In-gel digestionGel bands for mass spectrometric analysis were processed according to

Shevchenko et al. (2006). Gel pieces were washed with 100 mM NH4HCO3 andHPLC-grade acetonitrile (1:1, v/v) (buffer A). Proteins were in-gel reduced by10 mM DTT in buffer A, and subsequently alkylated with 55 mM iodoacetamidein buffer A. Proteins were digested overnight at 37 �C in digestion buffer (40 mMNH4HCO3, 10% acetonitrile) containing 12.5 ng/ll proteomics-grade trypsin.Peptides were extracted with 100 ll 2:1 (v/v) ACN: 5% FA. Extracts were dried ina vacuum centrifuge and reconstituted in 10 ll of mobile phase A (Section 2.8.3).

2.8.2. In-solution digestionSamples from Immunoprecipitation were reconstituted in 250 ll HPLC-grade

water, reduced with 10 mM DTT (1 h, 37 �C), alkylated with 24 mM iodoacetamide(1 h, 37 �C) and digested with 600 ng proteomics-grade trypsin after quenchingwith 2 mM DTT (20 min, 37 �C). Peptides were purified by strong-cation exchangestage tips and subsequently injected for mass spectrometric analysis.

366 K.C.M. Verhoeckx et al. / Food and Chemical Toxicology 65 (2014) 364–373

2.8.3. Nano LC-MS/MS and database searchChromatography was performed on an Easy LC 1000 nanoscale liquid chroma-

tography (nanoLC) system (Thermo Fisher Scientific, Denmark). Four microlitrepeptide mixture was loaded at 500 nl/min directly onto a pulled silica capillary(75 lm i.d.), in-house packed to a length of 10 cm with 3 lm C18 silica particles(Dr. Maisch, Germany). For gel band analysis, gradient elution was achieved at350 nl/min flow rate, and ramped from 100% A (0.1% FA in 2% acetonitrile), to30% B (0.1% FA, 80% acetonitrile) in 15 min, then from 30% B to 100% B in 5 min.MS detection was performed on the Q-Exactive (Thermo Fisher Scientific, Germany)operating in positive ion mode, with nanoelectrospray (nESI) potential at 1800 V.Data-dependent acquisition was performed using a top-5 method. Mass windowfor precursor I on isolation was 2.0 m/z, whereas normalised collision energy was30. For in-solution digests, the following parameters were changed with respectto the gel spot method: (i) nanoLC gradient was ramped from 100% A to 60% A in100 min. Data were processed by Proteome Discoverer 1.3 (Thermo Fisher Scien-tific, Germany), using Sequest as search engine and the Swiss Prot database ac-cessed on February 2013 as sequence database. The following search parameterswere used: MS tolerance 7 ppm; MS/MS tolerance 0.02 Da; fixed modificationscarbamidomethyl cysteine; enzyme trypsin; max. missed cleavages 2; taxonomyMetazoa (for gel bands) or Insecta (for immunoprecipitated extracts). Protein hitsbased on two successful peptide identifications (Xcorr > 2.0 for doubly charged pep-tides, >2.5 for triply charged peptides, and >3.0 for peptides having a charge state>3) were considered valid.

2.9. Indirect basophil activation test (BAT)

BAT was performed as described by Koppelman et al. (2004) with minor adap-tations. Peripheral blood mononuclear cells (PBMC) were isolated using density gra-dient centrifugation with Ficoll (Amersham, Sweden). Two mililitre of lactic acidwas used to strip basophils of IgE. Basophils were reloaded with specific IgE in incu-bation buffer with 15% patient serum. Cells (1 � 106) were incubated with equalvolumes of (i) RPMI (with 2 ng ml�1 IL-3 (R&D systems, USA) and 1% HSA) as neg-ative control; (ii) a 10-fold serial dilution of allergen (from 100 lg ml�1 to1 ng ml�1); or (iii) goat anti-human IgE antibody (Kirkegaard & Perry Laboratories,USA) as a positive control (3 lg ml�1, 1 lg ml�1 and 0.1 lg ml�1). Cells were subse-quently incubated in the dark (30 min, 37 �C). Activation was stopped with 25 ll icecold PBS plus 20 mM EDTA after which the cells were incubated in the dark (30 min,4 �C) with fluorescent labelled antibodies against CD63, CD123 and CD203c (Bioleg-end, USA). CD63, CD123 and CD203c expression was analysed by flow cytometryusing a FACSCanto II (BD Biosciences, USA), and FACSDiva software. Basophils wereidentified on forward-side scatter histograms as CD203c+CD123+ and activatedbasophils as CD203c+CD63+. The cut-off percentage for positive basophil activationwas determined as two times the percentage of activated basophils observed forRPMI + IL3 only.

2.10. Simulated gastric fluid digestion

Protein extracts (5 mg) were suspended in 10 ml water containing 23 mM citricacid and 0.38 mM Na2HCO3. The pH of the solution was set at 2.5. After incubation(5 min, 37 �C) T = 0 sample was collected (150 ll) and 900 units of porcine pepsin(1:0.07 protein:pepsin) was added. Digest samples were collected at 15 and 30 s,and at 1, 5, 10, 30, and 60 min and analysed using immunoblotting with sera frompatient 1.

3. Results

3.1. Yellow mealworm is taxonomically related to crustaceans andHouse dust mite (HDM)

Taxonomically, insects belong to the subphylum Hexapoda,which is one of the four subphyla of the phylum Arthropoda(Fig. 1). Within the arthropods, several pan-allergens are known,including tropomyosin (Reese et al., 1999), arginine kinase (Binderet al., 2001) and glutathione S-transferase (Galindo et al., 2001).For these allergens, cross-reactivity has been observed not only be-tween species within the same subphylum, but also between spe-cies from different arthropod subphyla, for example betweencrustacean species (e.g. shrimp, crab), chelicerates (e.g. mites)and several insects species (Binder et al., 2001; Galindo et al.,2001; Liu et al., 2009; Santos et al., 1999).

These findings suggest that people with a crustacean- or HDMallergy might experience allergic reactions upon consumption ofYellow mealworm.

3.2. Yellow mealworm extracts contain various putative allergens

Highly abundant proteins in the Yellow mealworm extractsSRN1 + 2 (water soluble) and SRN3 (urea soluble) were identifiedusing LC-MS/MS (Table 1). Only a few proteins were identified asspecific for Yellow mealworm (a-amylase and larval- and pupalcuticle proteins), due to the absence of a good database for Yellowmealworm proteins. Therefore, most proteins were identifiedbased on homology with other metazoan species.

Proteins identified in both water soluble extracts were highlysimilar (Table 1). Identified proteins which are also known aller-gens in other species are cationic trypsin (e.g. mites), arginine ki-nase (mites, crustaceans, insects), ovalbumin-like protein(chicken eggs), a-tubulin (mites) and a-amylase (mites, insects).For the urea soluble extract these were cationic trypsin, ovalbu-min-like protein and tropomyosin (mites, crustaceans, insects).Based on the above-mentioned results as well as the taxonomicrelationship (Fig. 1), sera from crustacean and/or HDM allergic pa-tients were selected for cross-reactivity studies (Table 2). In addi-tion, sera from food allergic patients not allergic to crustaceansor HDM Der p 10 were tested.

3.3. Allergic potential of Yellow mealworm proteins

The allergenic potential of identified Yellow mealworm proteinswith a known sequence, as well as all Yellow mealworm proteinsin the UniProt database was predicted using Allermatch™. A pro-tein can be considered potentially allergenic when it shows morethan 35% identity with a known allergen within a window of 80amino acids or more. For example, 392 hits >35% between Yellowmealworm a-amylase and Eur m 4 allergen indicates that for Yel-low mealworm a-amylase, Allermatch™ found 392 times >35% se-quence identity within 80 amino acid windows with Eur m 4.Potentially allergenic proteins based on above-mentioned criteriaare listed in Table 3.

3.4. Proteins in Yellow mealworm extract cross-react with IgE fromHDM and crustacean allergic patients

On immunoblot, six out of seven sera from HDM Der p 10- andcrustacean allergic patients showed IgE binding to all Yellow meal-worm extracts (Table 4 and Fig. 2). IgE binding was observed forprotein bands with MW between 25 and 40 kDa in both the watersoluble fractions and the urea soluble fraction. Serum of patient 3showed no IgE binding to any of the extracts tested, which could beexplained by lower IgE titers in the serum. Fifteen sera from pa-tients with an allergy to codfish, egg, milk, peanut or grass pollenand no allergy to HDM Der p 10 and crustaceans were also tested.These sera showed IgE binding to their respective controls, but notto proteins in Yellow mealworm extracts. A representative blot isshown in Fig. 2.

To determine whether cross reactivity observed in the immuno-blots was functional, five sera from crustacean/HDM Der p 10 aller-gic patients showing the strongest IgE binding to Yellowmealworm extracts were included in the indirect basophil activa-tion test (BAT). Positive basophil activation in response to Yellowmealworm extracts and shrimp extract was observed for all testedsera (Table 4). Sera from a grass pollen-, peanut- and a fish allergicpatient were used as negative controls. Fig. 2 shows three repre-sentative BAT results of one crustacean/HDM Der p 10 allergic pa-tient (patient 1) who reacted strongly to urea soluble extract andone crustacean/HDM Der p 10 allergic patient who reacted morestrongly to water soluble extract (patient 5). Patient 16 is a peanutallergic patient who did not react to any of the Yellow mealwormextracts, but did react to peanut extract. These results are repre-sentative for the other sera tested.

Fig. 1. Simplified representation of the phylogeny of the phylum Arthropoda, based on Regier et al. (2010), Rota-Stabelli et al. (2011).

Table 1Proteins identified in Yellow mealworm protein extracts.

Protein Accession Score Queries matched Mass (kDa) Sequence coverage (%)

Water soluble fractionsSRN1

Cationic trypsin P00760 554 16 26.5 45Calcium-transporting ATPase sarcoplasmic/ER type Q292Q0 511 15 109.9 11Arginine kinase P48610 438 11 40.1 17Actin P49871 366 15 42.1 28POTE ankyrin domain family member F A5A3E0 248 9 123.0 6Tubulin a-1 chain P06603 219 6 50.6 12Catalase P00432 212 3 60.1 3Alpha-amylase P56634 196 7 51.7 16Alpha-actinin P18091 161 6 107.6 6Muscle-specific protein 20 P14318 135 2 20.3 7Glyceraldehyde-3-phosphate dehydrogenase Q28259 127 4 36.1 8Ovalbumin-like P01012 127 2 43.2 8Tubulin b chain P41386 119 5 38.6 14Elongation factor 2 P13060 116 2 95.4 3

SRN2Cationic trypsin P00760 496 12 26.5 34Alpha-amylase P56634 370 10 51.7 20Arginine kinase P48610 344 9 40.1 14Calcium-transporting ATPase sarcoplasmic/ER type Q292Q0 316 8 109.9 7Actin P49871 313 13 42.1 23ATP synthase subunit b Q05825 286 6 54.1 814-3-3 protein zeta Q1HR36 280 4 28.3 21POTE ankyrin domain family member E Q6S8J3 225 8 122.9 7Catalase P00432 224 3 60.1 3Alpha-actinin P18091 153 5 107.6 4Muscle-specific protein 20 P14318 121 2 20.3 7

Urea soluble fraction (SRN3)Myosin heavy chain P05661 2115 71 225.4 20Actin P83969 517 27 42.1 33Cationic trypsin P00760 348 11 26.5 20Larval cuticle protein F1 Q9TXD9 335 7 14.9 51Larval cuticle protein A1A P80681 237 8 17.7 33Late histone H2A P16886 201 2 13.4 18Tropomyosin-1 Q1HPU0 200 5 32.6 16Pupal cuticle protein G1A P80685 192 4 20.8 11Ovalbumin-like P01012 191 3 43.2 8Tropomyosin-2 Q1HPQ0 154 10 32.8 23Myosin-2 P12845 134 3 223.0 1

Proteins identified by LC-MS/MS with a score of 100 and higher found in the water soluble protein fraction (SRN1), water soluble dialyzed protein fraction SRN2 and in theurea soluble protein fraction (SRN3). Identification was based on homology with metazoan proteins in Swiss Prot database. Putative allergens are listed in bold.

K.C.M. Verhoeckx et al. / Food and Chemical Toxicology 65 (2014) 364–373 367

3.5. Arginine kinase and tropomyosin were identified as major crossreactive allergens in Yellow mealworm

Cross-reactive proteins were identified using LC-MS/MS in twoways: (1) excision of bands from SDS–PAGE gels (Fig. 3, Table 5)

and (2) immunoprecipitation with IgE from crustacean/HDM Derp 10-allergic patients (Table 6). In most cases, more than one pro-tein was identified in the samples.

In both water soluble extracts (SRN1 and 2), arginine kinase wasidentified. In addition, actin was identified in the undialysed

Table 2Sensitisation pattern of the different patients tested in this study.

Patient Othera arthropods HDM HDMb Fish Milkc Eggd Pollene Peanutf PR-10g

Tropomyosin Tropomyosin Parvalbumin Food

Shrimp/Lobster/HDM allergyPatient 1 3 3 3 3 1 0 3 0 3Patient 2 3 3 3 0 0 0 3 0 1Patient 3 2 2 0 1 0 0 3 3 1Patient 4 3 3 3 0 2 2 3 3 3Patient 5 3 3 3 0 2 2 3 0 3Patient 6 2 2 0 0 2 3 3 3 2Patient 7 2 2 3 2 1 1 3 3 3

Grass pollen + food allergyPatient 8 0 0 0 1 0 0 3 0 1Patient 9 0 0 0 1 0 0 3 0 3Patient 10 0 0 0 0 0 0 3 0 0

Grass pollen without food allergyPatient 11 0 0 0 1 0 0 3 1 0Patient 12 0 0 0 1 0 0 3 0 0Patient 13 0 0 0 0 0 0 3 2 0

Peanut allergyPatient 14 0 0 1 0 0 0 0 2 0Patient 15 0 0 0 0 0 0 0 2 0Patient 16 0 0 1 0 0 0 1 3 0

Fish allergyPatient 18 0 0 3 2 0 0 2 0 1Patient 19 0 0 3 3 0 0 3 0 3Patient 20 0 0 0 2 0 0 0 0 0

Egg/milk allergyPatient 21 0 0 0 0 1 2 3 0 3Patient 22 0 0 3 0 2 2 3 0 2Patient 23 0 0 0 0 2 0 0 0 0

IgE antibody levels correspond to ISAC Standardised Units (ISU) as follows: 0 (undetectable of very low, <0.3 ISU); 1 (low, P0.3–<1 ISU); 2 (moderate to high, P1–<15 ISU); 3(very high. P15ISU).

a Pen a 1, Pen i 1, Pen m 1, Bla g 7, Ani s 3.b Der p 1, Der p 2, Der f 1, Der f 2, Eur m 2.c b-lactoglobulin, casein, lactoferrin.d ovomucoid, ovalbumin.e Cyn d 1, Phl p 1, Phl p 2, Phl p 4, Phl p 5, Phl p 6, Phl p 11, Phl p 12, Bet v 1, Aln g 1, Cor a 1.0101.f Ara h 1, Ara h 2, Ara h 3.g Ara h 8, Cor a 1.0401, Act d 8, Api g 1, Dau c 1, Gly m 4, Mal d 1, Pru p 1.

Table 3Potentially allergenic Yellow mealworm proteins according to Allermatch™. Only Yellow mealworm proteins with a known sequence were analysed.

Protein Sequence identity in Allermatch™

Allergen 80 AA sliding window analysis Full sequence alignment

# hits >35% identity % hits >35% identity % Overlap (AA) E

Alpha-amylase (P56634) Eur m 4 392 100.00 50.20 494 1.0e�68

Der p 4 392 100.00 49.70 493 2.7e�68

Putative trypsin-like proteinase (A1XG56) Blo t 3 179 100.00 47.47 257 4.2e�46

Eur m 3 162 90.50 46.36 220 1.9e�39

Der p 3 150 83.80 46.36 220 2.6e�39

Der f 3 120 67.04 44.09 220 1.4e�38

Tyr p 3 117 65.36 40.15 264 7.2e�35

Der p 9 109 60.89 40.44 225 4.0e�29

Der f 6 99 55.31 36.68 229 2.8e�22

Putative serine proteinase truncated (A1XG64) Blo t 3 21 80.77 39.39 99 1.0e�09

Der f 6 18 69.23 42.65 68 2.8e�08

Der p 9 13 50.00 42.03 69 1.3e�07

Cockroach allergen-like protein (Q7YZB8) Bla g 1 340 65.89 35.92 412 1.5e�42

Per a 1 259 50.15 35.59 413 4.0e�40

Expect value (E) indicates the number of hits one can expect to see by chance when searching a database of a particular size. Eur m (Euroglyphus maynei); Der p(Dermatophagoides pteronyssinus); Blo t (Blomia tropicalis); Der f (Dermatophagoides farinae); Tyr p (Tyrophagus putrescentiae); Bla g (Blatella germanica); Per a (Periplanetaamericana).

368 K.C.M. Verhoeckx et al. / Food and Chemical Toxicology 65 (2014) 364–373

fraction (SRN1) while fructose-biphosphate aldolase was identifiedin the dialysed fraction (SRN2). In the urea soluble protein fraction(SRN3), actin and tropomyosin were identified.

Immunoprecipitation of Yellow mealworm protein extractswas performed to identify more cross-reactive proteins includingthose that could not be identified by immunoblotting. More

Table 4Results of immunoblots (Blot) and indirect basophil activation tests (BAT) using crustacean/HDM Der p 10-allergic patient sera and Yellow mealworm protein extracts.

Patients BlotSRN1

BlotSRN2

BlotSRN3

Blottropomyosin

Blotshrimp/lobster

BATSRN1

BATSRN2

BATSRN3

BATtropomyosin

BATshrimp/lobster

Patient 1 + + + + + + + + + +Patient 2 + + + + + + + + + +Patient 3 � � � � � NT NT NT NT NT

Patient 4 + + + + + + + + + +Patient 5 + + + + + + + ± ± +Patient 6 + + ± ± + + + + ± ±Patient 7 ± ± + + + NT NT NT NT NT

Response was identified as positive response (+), mild (±) or no response (–) to Yellow mealworm extracts (water soluble; SRN1, water soluble dialysed; SRN2, urea soluble;SRN3), shrimp/lobster extract and tropomyosin. NT = not tested.

Fig. 2. Cross-reactivity shown by immunoblot (above) and indirect basophil activation test (below) of Yellow mealworm extracts with sera from crustacean/HDM Der p 10-allergic patients (patient 1 and 5) and a peanut-allergic patient, not allergic to crustaceans or HDM Der p 10 (patient 16). These results are representative for other patient seratested (n = 7 crustacean/HDM Der p 10-allergic and n = 15 allergic to either codfish, egg, milk, peanut or grass pollen) SRN1 = water soluble protein; SRN2 = water soluble,dialysed protein; SRN3 = urea soluble protein; S/L = shrimp/lobster extract; CPE = crude peanut extract.

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putative allergens were found by immunoprecipitation. The topfive based on identification score and number of identified pep-tides is listed in Table 6, in addition to identified proteins knownto be allergenic in other species. For the water soluble fractions,arginine kinase was identified with the highest score, corre-sponding with the results of protein band identification. Actinwas also identified using both techniques. Fructose-biphosphatealdolase was not identified within the top five. For the ureasoluble extract, both actin and tropomyosin were identifiedusing both techniques. In addition, a troponin-T -like protein,a- and b-tubulin and a light chain myosin-like protein wereidentified.

3.6. Cross-reactive proteins were moderately stable

Proteins in the water soluble fractions were partly digestedafter 15 s, after which they stayed relatively stable until 10 min.

After 30 min, proteins were further digested. Digestion was,however, not completed after 60 min and the fragments could stillbind IgE. In the urea soluble fraction, protein bands of approxi-mately 32 kDa were completely digested after 10 min, while a pro-tein of approximately 40 kDa followed the same kinetics as theproteins in the water soluble fractions (Fig. 4).

4. Discussion

In this study, the weight of evidence approach as described bythe European Food Safety Authority (EFSA) (EFSA, 2010), was usedto assess the allergenic potency of Yellow mealworm proteins. Tothis end, proteins were identified in Yellow mealworm extract.The allergic potential of Yellow mealworm proteins with a knownsequence was determined. Cross-reactivity of Yellow mealwormproteins with IgE from crustacean and House dust mite (HDM)Der p 10 allergic patients was assessed in vitro and the stability

Fig. 3. Yellow mealworm protein bands showing cross-reactivity in immunoblot (left) excised from Coomassie-stained gel (right). SRN1 = water soluble protein;SRN2 = water soluble, dialyzed protein; SRN3 = urea soluble protein; S/L = shrimp/lobster extract. Numbers of bands correspond with Table 5.

Table 5Yellow mealworm proteins identified in excised bands using LC-MS/MS and metazoan database.

Band Protein LC-MS/MS analysis Theoretical

Accession Score Mass (kDa) No. of peptides (unique/matched) Sequence coverage (%)

SRN11 Arginine kinase Q9U9J4 244 40.6 2/8 22

Actin, muscle P49871 149 42.1 0/6 192 Actin O16808 101 42.1 1/4 19

Arginine kinase Q9U9J4 100 40.6 2/4 9

SRN23 Arginine kinase Q9U9J4 242 40.6 2/8 22

Fructose-biphosphate aldolase P07764 56 39.3 1/1 24 Fructose-biphosphate aldolase P07764 55 39.3 1/1 2

Arginine kinase Q9U9J4 46 40.6 1/1 25 Arginine kinase P48610 112 40.1 1/4 16

SRN36 Tropomyosin Q9NG56 54 32.9 0/2 87 Tropomyosin O96764 39 32.6 0/1 48 Actin, indirect flight muscle P83969 195 42.1 0/6 19

Tropomyosin-1 isoforms 9A/A/B P06754 89 39.4 0/4 109 Actin, muscle P49871 120 42.1 0/6 19

Tropomyosin-1 isoforms 9A/A/B P06754 89 39.4 0/4 10

SRN1 = water soluble fraction, SRN2 = water soluble dialysed fraction, SRN3 = urea soluble fraction.

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of the cross-reactive proteins was determined in a static pepsindigestion test.

Proteins in the mealworm extracts were identified based onhomology with other metazoan species, since only a few proteinsequences from the Yellow mealworm are available in the proteindatabase. The water soluble fractions contained putative allergenssuch as cationic trypsin, arginine kinase, a-tubulin, a-amylase andan ovalbumin-like protein. The urea soluble fraction contained theputative allergens cationic trypsin, tropomyosin and an ovalbu-min-like protein. Arginine kinase is an enzyme most often presentin invertebrates and allergic cross-reactivity has been observedbetween different crustaceans (e.g. Black tiger shrimp pen m 2,Penaeus monodon; White shrimp Lit v 2, Litopenaeus vannamei;Mud crab Scy pa 2, Scylla paramamosain), HDM (Der p 20) andinsect species such as Indian meal moth (Plo i 1, Plodiainterpunctella), Domesticated silkworm (Bomb m 1, Bombyx mori)and two cockroach species (Bla g 9, Blatella germanica; Per a 9,Periplaneta americana) (Binder et al., 2001; Liu et al., 2009; Yu

et al., 2013). Trypsin-like enzymes were described as HDMallergens Der f 3 en Der p 3 (Ando et al., 1993; Steward et al.,1992). Alpha-tubulin has been described as an allergen in storagemites Tyrophagus petruscentiae and Lepidoglyphus destructor (Jeonget al., 2005; Saarne et al., 2003) while the enzyme a-amylase isknown as HDM allergens Blo t 4 (Blomia tropicalis), Der p 4(Dermatophagoides pteronyssinus), and Eur m 4 (Euroglyphusmaynei) (Thomas et al., 2010; Yan Chua et al., 2007). Tropomyosinsare highly conserved key regulatory proteins involved in the con-traction of muscle and non-muscle cells (Behrmann et al., 2012).Cross-reactivity has been observed between tropomyosins fromcrustaceans, mites and several insect species (Santos et al., 1999).While being a major crustacean allergen (Gámez et al., 2011),tropomyosin is a minor allergen in HDM. The dominant HDMallergens, Der p 1 and Der p 2, are recognised by over 90% ofHDM allergic patients (Bronnert et al., 2012; Weghofer et al.,2005) while only 5–15% of patients recognise Der p 10 (Asturiaset al., 1998; Resch et al., 2011). However, patients with both a

Table 6Top five proteins based on score and proteins known to be allergenic (bold) identified in immunoprecipitation exacts of Yellow mealworm.

Protein LC-MS/MS analysis Theoretical

Accession Score Mass (kDa) No. of peptides(unique/matched)

Sequence coverage (%)

Bead 1: SRN1 + SRN 2 with serum patients 2, 5 and 6Arginine kinase D9YT56 38.49 39.6 2/5 16.06Myosin heavy-chain like D6WVJ3 35.23 262.1 15/15 7.13Titin-like D6WIF5 15.52 2048.6 7/7 0.44Actin-87E B0WEY5 13.53 41.8 4/4 13.83Limpet-like D6WJL5 13.40 46.5 5/5 13.55

Beta-tubulin Q1PC35 10.31 26.8 3/3 13.19Alpha-amylase P56634 6.67 51.2 3/3 6.79Tropomyosin-like D6X4X2 6.26 75.2 2/2 3.37Paramyosin I4DIM8 5.48 102.3 3/3 3.77Alpha-tubulin (fragment) I6T3A6 4.83 35.0 2/2 6.07Cockroach allergen-like protein Q7YZB8 4.59 65.4 2/2 6.72Glutathione S-transferase-like D6WH21 2.00 23.6 2/2 9.31

Bead 2: SRN3 with serum patient 1Myosin heavy chain-like D6WVJ3 975.94 262.1 28/85 29.56Titin-like D6WIF5 286.63 2048.6 53/62 4.45Actin, muscle E2B152 242.60 41.7 3/14 39.10Troponin T-like D6W953 154.25 45.7 15/15 23.30Tropomyosin-like D6X4X3 117.99 32.3 7/14 35.56

Paramyosin-like J3JWD1 75.20 101.9 5/9 10.34Arginine kinase (fragment) D5L6P4 26.54 27.0 4/6 31.51Heat shock protein 70 D2Y0Z5 21.94 71.0 4/6 12.02Chitinase Q8MP05 20.78 321.2 8/8 3.56Troponin C-like D6WZP8 16.72 17.5 3/6 20.39Beta-tubulin-like H9JHY3 14.38 45.5 5/5 13.73

Heat shock protein 90-like K7IS89 12.05 82.0 3/4 16.34Alpha-tubulin (fragment) I6TYI6 10.71 41.4 4/4 5.17Alpha-amylase P56634 6.96 51.2 3/3 14.59Myosin light chain-like D6WZU7 5.56 31.3 2/2 6.79

Identification was based on homology with known insect sequences. High scores indicate high probability of protein/peptide identification. SRN1 = water soluble fraction,SRN2 = water soluble dialysed fraction, SRN3 = urea soluble fraction.

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HDM and crustacean allergy express higher levels of IgE to Der p 10than to the major HDM allergens (Bronnert et al., 2012). This wasalso the case for most patients in our database with an allergy tocrustacean tropomyosin.

All Yellow mealworm proteins with a known sequence in Uni-Prot database were analysed for allergic potential in Allermatch™.Proteins indicated to be potentially allergenic were a-amylase, aputative trypsin-like proteinase and a putative serine proteinase,which show sequence identity to several known mite allergens.Additionally, cockroach allergen-like protein shows sequence iden-tity to cockroach allergens and was indicated to be potentiallyallergenic. Several known pan-allergens which were identified inYellow mealworm extracts, such as tropomyosin and argininekinase, could not be analysed in Allermatch™ because at thismoment no sequence information is available for these proteinsfrom Yellow mealworm.

Choice of patient sera was based on arthropod phylogeny andproteins identified in Yellow mealworm extracts. All Yellow meal-worm extracts showed cross-reactivity with sera from crustacean/HDM Der p 10 allergic patients on immunoblot. Cross-reactivity ofYellow mealworm proteins with IgE from crustacean/HDM Der p10-allergic patients was proven functional in indirect BAT. Degran-ulation was between 10% and 40%, depending on the sera tested.The positive control (shrimp/lobster extract) showed degranula-tion results comparable to Yellow mealworm extracts. Low degran-ulation in response to Yellow mealworm extracts still exceededbackground degranulation (two times the percentage of activatedbasophils observed for RPMI + IL3 only, results not shown). Theobserved cross-reactivity was expected, because both fractionscontain proteins (e.g. tropomyosin and arginine kinase), whichare important cross-reacting arthropod pan-allergens (Binder

et al., 2001; Reese et al., 1999). Sequence identities between theseallergens in arthropods were at least 65% (Barletta et al., 2005;Santos et al., 1999).

LC-MS/MS analysis identified the major cross-reactive Yellowmealworm proteins as arginine kinase in the water soluble fractionand tropomyosin in the urea soluble fraction. In addition, actin wasrevealed as cross-reactive protein. Actin is not known as an allergenin arthropods. Sequence analysis of identified actins in Allermatch™did not show sequence similarity to known allergens (results notshown). However, actin binds strongly to tropomyosin, forming acomplex which is essential in muscle contraction (Behrmann et al.,2012). It is possible that identification of actin was caused by immu-noprecipitation of the tropomyosin–actin complex. In addition, ac-tin co-elutes with tropomyosin on SDS–PAGE gel. Insect actinsmight be putative allergens, but this remains to be proven. Severalother allergens were identified, such as troponin T, tubulins and lightchain myosin. Tubulins and light chain myosin are known arthropodallergens (Ayuso et al., 2008). It is unknown whether troponin Tcould act as an allergen.

Cross-reactive Yellow mealworm proteins were moderately sta-ble in the static pepsin digestion model compared to Ara h 1, whichwas completely degraded within 15 s and Ara h 2, which was notcompletely digested even after 60 min using the same experimen-tal conditions (data not shown).

To our knowledge, this is the first study to determine cross-reactivity between Yellow mealworm proteins and IgE from crus-tacean/HDM Der p 10 allergic patients. Because Yellow mealwormis not commonly eaten yet, sensitisation to Yellow mealworm pro-tein through the oral route is unlikely for the tested patients.Unfortunately, sera from patients allergic to Yellow mealwormprotein are not readily available. It is therefore not possible to

Fig. 4. Digestion kinetics of Yellow mealworm extracts SRN1 (water soluble fraction), Spepsin digestion model shown on Coomassie-stained gel (above) and immunoblot (belo

372 K.C.M. Verhoeckx et al. / Food and Chemical Toxicology 65 (2014) 364–373

determine the risk of sensitisation to mealworm proteins yet.However, sensitisation is always a risk with novel proteins, andcross-reactivity can occur without direct sensitisation. This is illus-trated by Fernandes et al. (2003), who described IgE reactivity toshrimp tropomyosin in orthodox Jews who were allergic to house-hold allergens such as HDM and cockroach, but had no previousexposure to shellfish.

The results we presented in this paper indicate that crustacean/HDM Der p 10 allergic patients may experience an allergic reactionwhen consuming products containing Yellow mealworm protein.However, to confirm this, it is imperative to challenge patientswith Yellow mealworm extract by means of a double-blindplacebo-controlled food challenge, which is the gold standard inallergy diagnosis.

In conclusion, there is a realistic possibility that HDM- andcrustacean allergic patients may react to food containing Yellowmealworm proteins.

Conflict of Interest

The authors declare that there are no conflicts of interest.

Transparency Document

The Transparency document associated with this article can befound in the online version.

Acknowledgement

RN2 (water soluble, dialyzed fraction) and SRN3 (urea soluble fraction) in a staticw) with serum from a representative crustacean/HDM Der p 10 allergic patient (1).

The authors like to thank Kreca for providing insect material.The authors are member of Cost action INFOGEST FA1005.

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